# Dissecting the impact of RNA on DNA replication and chromatin structure

> **NIH NIH R35** · NEW YORK UNIVERSITY · 2020 · $550,793

## Abstract

PROJECT SUMMARY
Collisions between the DNA replication and transcription machineries (replication-transcription conflicts) appear
to be common in eukaryotic cells. Although these conflicts have long been studied as a potential source of DNA
damage and, therefore, a threat to genome integrity, we lack a detailed molecular understanding of how the
presence of transcribing RNA polymerases on DNA affects the progress of replication, and of the mechanism(s)
by which these replication-transcription conflicts give rise to DNA damage.
Another consequence of transcription during DNA replication is elevated levels of ribonucleoside triphosphates
(rNTPS) – the substrate for RNA polymerases. Due to incomplete discrimination between rNTPs and dNTPs by
the replicative DNA polymerases, large numbers of ribonucleotides are mis-incorporated into the genome during
each round of replication; this burden is estimated at > 1 million ribonucleotides per cell division in human cells,
making ribonucleotides by far the most abundant lesion in eukaryotic DNA. Mis-incorporated ribonucleotides are
removed via the ribonucleotide excision repair (RER) pathway, and impaired removal is linked to several human
diseases. However, it is not known how ribonucleotides impact chromatin – the higher-order structure of DNA –
or conversely how chromatin affects RER. Furthermore, it has not been determined whether all ribonucleotides
are equally amenable to repair or what may underlie differences in RER efficiency through the genome.
The proposed work encompasses two ongoing projects:
The first project addresses how orientation-dependent effects on replication progression and genome integrity
arise at transcribed genes. To achieve this, we use a recently developed quantitative method to assay the
movement of the replisome genome-wide at high resolution, in combination with genome-wide interrogation of
DNA double-strand break formation and a novel assay to map nascent DNA strands in the context of an arrested
replication fork.
The second project uses a combination of genome-wide assays and in vitro biochemistry to delineate how
ribonucleotides destabilize nucleosomes (the basic repeating unit of chromatin), how nucleosomes affect RER
initiation by the RNase H2 enzyme, and to elucidate the dynamics of RER at all loci in the genome.
The machineries responsible for DNA replication, transcription, and DNA repair are highly conserved throughout
eukaryotes. Both projects will be carried out in the budding yeast Saccharomyces cerevisiae: the small genome,
rapid replication, and genetic manipulability of S. cerevisiae make this an ideal model in which to study the
intersection of fundamental biological processes. Therefore, the results of this work will provide molecular
insights into genome instability in humans, and will be directly applicable to our understanding of the etiology
and progression of cancer as well as rare diseases including Aicardi-Goutières syndrome.

## Key facts

- **NIH application ID:** 9851570
- **Project number:** 1R35GM134918-01
- **Recipient organization:** NEW YORK UNIVERSITY
- **Principal Investigator:** Duncan J Smith
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $550,793
- **Award type:** 1
- **Project period:** 2020-03-01 → 2025-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9851570

## Citation

> US National Institutes of Health, RePORTER application 9851570, Dissecting the impact of RNA on DNA replication and chromatin structure (1R35GM134918-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9851570. Licensed CC0.

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